摘要
近年来,随着我国毛皮动物规模化养殖业的不断扩大,由病原细菌所引起的狐类感染,常表现出流行面广、发病率和死亡率高的特点。传染性疾病,尤其是细菌性疾病的多重感染发生日益频繁、越来越普遍,给毛皮动物养殖生产带来了较大的经济损失,严重地威胁到毛皮动物养殖业的持续发展。2006-2007年间,山东6个地区不同狐群养殖场养殖的母狐出现空怀、流产、出血性肺炎等症状病变类似的疫情。为了进一步探讨引起这场规模化狐场发生疾病的原因,本研究开展了流行病学调查,病原学检查,细菌多重感染的状况分析及间接荧光抗体检测等方法的建立,并对其主要病原进行16SrRNA序列测定和系统进化性分析,为规模化种狐场多重感染的疾病诊断与防治提供了理论依据。
本研究共分三部分:
一、规模化蓝狐养殖场细菌感染的病原学检测及其多重感染的状况分析
2006-2007年间,山东6个地区不同狐群养殖场养殖的母狐出现空怀、流产、出血性肺炎等症状病变类似的疫情,给养殖场造成了巨大经济损失。通过对其养殖场(共计饲养蓝狐2.2万余只)进行流行病学调查,对送检的236只病死蓝狐进行细菌和病毒学检查,及犬瘟热(CDV)、犬细小病毒(CPV)快速诊断试纸条的检测,从被检病料中主要分离到绿脓杆菌(86%)、大肠杆菌(34.3%)、沙门氏菌(25.8%)和普通变形杆菌(8.5%)四种病原菌,病毒检查与试纸条检测皆为阴性,人工感染小鼠的致病性试验结果表明四种病原菌皆为致病菌;通过细菌分离率及共感染率的统计分析,发现病狐群同时存在大肠杆菌、沙门氏菌、普通变形杆菌和绿脓杆菌的二重感染(45.8%)、三重感染(3.8%)、甚至四重感染(0.4%)的情况,尤其是以绿脓杆菌感染为主的多重感染,已成为影响山东地区毛皮动物养殖疫病的主要病因之一。
二、蓝狐绿脓杆菌主要血清学诊断方法的建立与临床应用
取上述试验所分离的主要病原菌绿脓杆菌的代表菌株ZB4,分别制备全菌(OK)抗原及菌体(O)抗原,经分别强化免疫接种家兔制备高效价抗血清,进行与待检菌株的玻片凝集反应、免疫荧光抗体反应(IFA)及琼脂扩散试验(AGP)等方面的免疫检验,结果在同种细菌间的荧光抗体检验显示出了特异的黄绿色荧光反应;玻片凝集反应中出现了特异凝集;在琼脂扩散检验中出现特异的免疫沉淀线。本研究建立了蓝狐绿脓杆菌玻板凝集、IFA、AGP的快速诊断方法,补充了传统细菌学检测的方法,为这些病原的快速、准确诊断奠定了基础。
三、蓝狐绿脓杆菌ZB4株16SrRNA基因的序列测定及系统进化分析
病原学研究结果表明绿脓杆菌是影响规模化蓝狐养殖场多重感染的主要病原之一,为进一步研究其分子生物学特性,本试验利用原核生物16SrRNA基因通用引物对分离纯化的蓝狐致病性绿脓杆菌菌株ZB4进行16SrRNA基因的克隆及序列分析。采用DNAstar软件将获得的序列与选取的核苷酸同源性较高的序列进行同源性比对,采用MEGA(3.1)软件生成同源序列的进化树。结果显示该菌株与假单胞菌属的9个代表菌株的同源性均很高,在98.7%～99.2%之间,在所构建的系统进化树上,ZB4菌株与绿脓杆菌(Pseudomonas aeruginosa)(AJ249451)的距离最近,位于同一分支,其同源性达99.2%。从分子水平上鉴定菌株ZB4为绿脓杆菌(Pseudomonas aeruginosa)。
In recent years, for large-scaled feeding industry’s unceasing expansion of fur animals, the infection of fox often shows major epidemic areas, higher morbidity and mortality, infectious diseases especially the multiple infection caused by bacteria occurred increasingly frequent, more and more popular, resulted in a great economic loss and threatened the feeding industry’s persistent development of fur animals acutely. From 2006 to 2007, the foxes in different fox farms in 6 areas of Shandong showed non-pregnancy, abortion, hemorrhagic lung fever etc. In order to approach the reasons of the diseases in the large-scaled farms further, the methods of epidemiologic survey, etiologic survey, the analysis of bacterial multiple infectious situation and the detection of fluorescent antibody were established in this research, we made a sequencing of 16SrRNA and analysis of the phyletic evolution for the major etiological agent and provided a theoretical basis in diagnosing disease and prevention and cure for multiple infections in scaled fox farms.
The research divides three parts:
Part One: The etiologic survey and analysis of multiple infectious situation of the bacterium in large-scaled alopex farms
From 2006 to 2007, the foxes in different fox farms in 6 areas of Shandong showed non-pregnancy, abortion, hemorrhagic lung fever etc, resulted in a great economic loss to the farms. By the epidemiologic survey in the farms and the etiologic survey of the 236 dead alopexs collected, four major pathogenic bacteria: aeruginosus bacillus (86%), bacillus coli (34.3%), c (25.8%) and bacillus proteus (8.5%); By the statistical analysis of the infectious state, we can find that there has dual infection, triple infection, and even quartet infection of bacillus coli, bacillus coli, bacillus proteus and aeruginosus bacillus in a considerable proportion of the alopex groups, especially the multiple infection of aeruginosus bacillus, and became a major etiological factor of the fur animals’loimia influenced Shandong.
Part Two: The establishment of major serodiagnosis method and clinical application of aeruginosus bacillus in alopex
Using the representative strain of the major pathogenic bacteria, aeruginosus bacillus, from the experiment above-mentioned, prepared whole-cell antigen and thallus antigen respectively and prepared antiserum corresponding by immunizing rabbits respectively, we can do the inspection such as immune serologic microscopic agglutination, immunofluorescent antibody response and agar diffusing response and so on by using the corresponding bacterial antiserum and unknown strains, the result is that the appearance of idio-fluorescence response of the fluorescent antibody inspection among homogeneous bacterial; the appearance of the specific immune precipitation line in agar-diffusing inspection. This research indicated the feasibility of inspecting the inspected bacteria and became a beneficial supplement of traditional bacterial identity, providing a reliable method for diagnosing the disease quickly and accurately.
Part Three: The ZB4 strain’s genic sequencing of 16SrRNA and analysis of the phyletic evolution of alopex’s aeruginosus bacillus
The result of aetiological study indicated that aeruginosus bacillus is one of the major reasons which influenced the multiple infections of the large-scaled alopex farms, in order to investigate the characteristics of molecular biology further, the consensus primer of 16SrRNA genes of procaryote were used to clone the 16SrRNA genes of alopex’s pathogenic aeruginosus bacillus isolated and purified and analyze sequence. The DNAstar software was used to analyze the homology between the sequence acquired and the sequence had a high homology with the sequence selected and the MEGA(3.1) software was used to form analogs’cladogram. The result indicated that there’s a high homology, from 98.7% to 99.2%, between the bacterial strain and nine representative strains of Pseudomonas, through phylogenetic trees constructed, the ZB4 strain and Pseudomonas aeruginosa (AJ249451) had a nearest evolutionary distance and the homology was 99.2%.
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